Nucleic acid extraction from formalin-fixed paraffin-embedded cancer cell line samples: a trade off between quantity and quality?

Background: Advanced genomic techniques such as Next-Generation-Sequencing (NGS) and gene expression profiling, including NanoString, are vital for the development of personalised medicines, as they enable molecular disease classification. This has become increasingly important in the treatment of cancer, aiding patient selection. However, it requires efficient nucleic acid extraction often from formalin-fixed paraffin-embedded tissue (FFPE). Methods: Here we provide a comparison of several commercially available manual and automated methods for DNA and/or RNA extraction from FFPE cancer cell line samples from Qiagen, life Technologies and Promega. Differing extraction geometric mean yields were evaluated across each of the kits tested, assessing dual DNA/RNA extraction vs. specialised single extraction, manual silica column based extraction techniques vs. automated magnetic bead based methods along with a comparison of subsequent nucleic acid purity methods, providing a full evaluation of nucleic acids isolated. Results: Out of the four RNA extraction kits evaluated the RNeasy FFPE kit, from Qiagen, gave superior geometric mean yields, whilst the Maxwell 16 automated method, from Promega, yielded the highest quality RNA by quantitative real time RT-PCR. Of the DNA extraction kits evaluated the PicoPure DNA kit, from Life Technologies, isolated 2–14× more DNA. A miniaturised qPCR assay was developed for DNA quantification and quality assessment. Conclusions: Careful consideration of an extraction kit is necessary dependent on quality or quantity of material required. Here we provide a flow diagram on the factors to consider when choosing an extraction kit as well as how to accurately quantify and QC the extracted material

Iron Behaving Badly: Inappropriate Iron Chelation as a Major Contributor to the Aetiology of Vascular and Other Progressive Inflammatory and Degenerative Diseases

The production of peroxide and superoxide is an inevitable consequence of aerobic metabolism, and while these particular "reactive oxygen species" (ROSs) can exhibit a number of biological effects, they are not of themselves excessively reactive and thus they are not especially damaging at physiological concentrations. However, their reactions with poorly liganded iron species can lead to the catalytic production of the very reactive and dangerous hydroxyl radical, which is exceptionally damaging, and a major cause of chronic inflammation. We review the considerable and wide-ranging evidence for the involvement of this combination of (su)peroxide and poorly liganded iron in a large number of physiological and indeed pathological processes and inflammatory disorders, especially those involving the progressive degradation of cellular and organismal performance. These diseases share a great many similarities and thus might be considered to have a common cause (i.e. iron-catalysed free radical and especially hydroxyl radical generation). The studies reviewed include those focused on a series of cardiovascular, metabolic and neurological diseases, where iron can be found at the sites of plaques and lesions, as well as studies showing the significance of iron to aging and longevity. The effective chelation of iron by natural or synthetic ligands is thus of major physiological (and potentially therapeutic) importance. As systems properties, we need to recognise that physiological observables have multiple molecular causes, and studying them in isolation leads to inconsistent patterns of apparent causality when it is the simultaneous combination of multiple factors that is responsible. This explains, for instance, the decidedly mixed effects of antioxidants that have been observed, etc...Comment: 159 pages, including 9 Figs and 2184 reference

Role of early trabeculectomy in primary open-angle glaucoma in the developing world.

BACKGROUND: The lack of prospective data comparing early surgery and medical management in primary open-angle glaucoma (POAG) in the developing world led us to conduct a small randomised controlled clinical trial to evaluate acceptance and effectiveness of early trabeculectomy in these patients. METHODS: A total of 60 patients with moderately advanced POAG were randomised into three groups (Group I-Conventional medical management, Group II-Option for early trabeculectomy, Group III-Received an educational package about their disease before an option for early trabeculectomy). The patients were followed up for a period of 6 months for visual acuity, intraocular pressures (IOP), and subjective satisfaction. RESULTS: The three study groups were statistically similar with respect to mean IOP, demographic, and socio-economic profile. 35% of the patients accepted early surgery when offered a choice between early surgery and medical management in one of the groups. 65% of patients in another group expressed willingness for an early surgery after receiving health education on glaucoma. The mean IOP in the operated eyes was lower than the medically treated eyes at 2 weeks (16.6 vs 23.0 mmHg), 6 months (18.5 vs 22.8 mmHg), and 1-year review (17.9 vs 22.3 mmHg) (P<0.001). No significant difference was seen among the groups with regard to visual acuity and subjective satisfaction. CONCLUSION: There is a reasonable acceptance of early surgery in POAG patients in the developing world and increases on educating patients about their disease. Early surgery offers better IOP control with no long-term subjective adverse effects